Biological molecules such as proteins, biopolymers, and enzymes including cytochrome c, catalase, ferriten, antibiotics, myoglobin, hemoglobin, and the like have been encapsulated into porous, inorganic host matrices, including transparent glasses and/or oxides (Bescher and Mackenzie, Mat. Sci. Eng., C6:145-154 (1998); Messing, Immobilized Enzymes for Industrial Reactors, Academic Press, New York (1975)). The metal alkoxide based process is the conventional choice for preparing an inorganic/organic hybrid at low temperatures (Klein, Annual Review of Materials Science, p. 227, Huggins et al., ed., Vol.15, (Palo Alto Calif.), 1985; Brinker and Scherer, Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing, Academic, Boston, 1990). The fragile characteristics of protein biomolecules limit the synthesis of inorganic/biomolecule composites to a sol-gel process for encapsulating the biological material into transparent silica matrices, which are capable of responding at a temperature significantly below the conventional sintering or melting temperature of the inorganics (Ellerby et al., Science 255:1113-15 (1992)); U.S. Pat. No. 5,200,334 ("Dunn et al. '334 patent")).
The basic concept of the sol-gel process involves hydrolysis and polymerization of inorganic monomers, typically using metal alkoxides, such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS). The biomolecules are added to and dispersed within the solution, to which a buffer solution has been introduced to adjust the solution pH to a level suitable for biomolecule survival. Hydrolysis of the alkoxide, e.g., TMOS, in the presence of water, an organic solvent and an acid/base catalyst results in the formation of --Si--OH bond. As the metal oxides undergo hydrolysis and polymerization reactions, the --Si--OH, silanol bonds further polymerize into --Si--O--Si-- bonds, and an inorganic three-dimensional network finally develops within a firm gel. As the gel dries, it shrinks and hardens further into an inorganic matrix (sometimes referred to as an aerogel) with a pore size typically in the range of 2-5 nm. The residue solvents are expelled from the growing network and the dispersed biomolecules are finally trapped into network cages (Dave et al., Ana. Chem. 66[22]:1120A-1127A (1994)). After a relatively long period of time, e.g., a few weeks, the solvent evaporates to result in a biomolecule-doped, transparent, dried matrix (Ellerby et al., 1992).
The biological molecules entrapped within the matrices reportedly retain high-levels of biological activity (Ellerby et a., 1992; Miller et al., J. Non-Crystalline Solids 202:279-289 (1996); Narang et aL, Ana. Chem 66[19]:3139 (1994); Livage et al., J. Sol-Gel Sci. Tech. 7:45-51 (1996); Coche-Guerente et at., Chem. Alater. 9: 1348-52 (1997); Kurokawa et al., Biotech. Bioengineer. 42:394-397 (1993); Glezer and Lev, J. Am. Chem. Soc. 115:2533-34 (1993); Tatsu et at., Chem. Letters 1615-1618 (1992); Lan et al., J. Sol-Gel Sci. Tech. 7:109-116 (1996)). Presumably, this is because the native-state conformation of the encapsulated molecule is sufficiently retained to permit a degree of measurable activity. Meanwhile, the matrix pores allow the diffusion of reactant molecules and their reaction with the entrapped biomolecules. Through these reactions, the biomolecule-containing inorganic monolith materials possess an ability to detect other molecules, and can thus be used as sensors for optical, electrical, mechanical or chemical signals (Bescher and Mackenzie,1998; Bakul et al., Ana. Chem. 66[22]:1121A (1994)).
Both the gel and aerogel are typically, to a certain extent, transparent to visible light and permeable to gases, wetting agents, ions and small molecules. Thus, the structure and properties of encapsulated biomolecules can be examined via spectroscopic means. Unfortunately, however, although reports have indicated the successful retention of up to 80% of the protein activity, the presence of alcohol in these alkoxide-based sol-gel processes has been known to denature proteins, thereby causing chain unfolding, aggregation, and destruction of secondary and tertiary protein structures to a significant extent (Miller et al., J. Non-Crystalline Solids 202:279-289 (1996)).
Alcohol is known to be lethal to many higher level biomolecules that have more delicate structures and more complicated functions, and it may impair the activity of other biomolecules to some extent even when they survive. Although the TMOS method by Ellerby et al., 1992 and the Dunn et al. '334 patent claim to use a "non-alcoholic medium," and teach that the addition of alcohol is not necessary to form the sol, any use of a metal alkoxide will release alcohol during hydrolysis. For example, in the case of TMOS, methanol is released according to the following reaction: Si(OMe).sub.4 +H.sub.2 O.fwdarw.Si(OMe).sub.4- +(OH).sub.x +x CH.sub.3 OH.
In the preceding reaction, Me represents the CH.sub.3 group. The methanol (CH.sub.3 OH) content, x, increases with the extent of hydrolysis. When hydrolysis is complete, a maximum of 4 moles of methanol is released for every mole of TMOS hydrolyzed. Since hydrolysis is the basis of the TMOS sol-gel method taught by Ellerby et al., 1992 and by the Dunn et al.'334 patent, alcohol always exists in the composite once the hydrolysis reaction begins. This is easily verified by the smell of alcohol that always accompanies any product of an alkoxide reaction, even though no alcohol has been expressly added.
The damaging effect of alcohol in the process is clear. For example, Miller et al., 1996, have reported a 70% reduction of the enzymatic activity when bovine liver catalase was encapsulated into a TMOS-based silica matrix, with merely 5 vol% methanol present. Moreover, a number of spectroscopic studies on protein denaturation have found an altered intensity and longwave shift of the absorption spectra when polar perturbing solvents, such as alcohols, interact with the aromatic amino acid residues of the proteins (Herkovits and Sorensen, Biochemistry, 7:2523-2533 and 2533-2542 (1968); Izumi and Inoue, J. Biochem. (Tokyo), 79, 1309-1321 (1976); Timasheff and Inoue, Biochemistry, 7:2501-2513 (1968); and Strickland et al., Biochemistry 11:3657-3662 (1972)).
Accordingly, until the present invention, there has been a long-felt need in the art for the development of an alcohol-free, sol-gel process for the encapsulation of active, biological materials in a visually transparent porous, inorganic matrix, thereby preventing the denaturation of biomolecule caused by the undesirable interaction with the alcohol molecules and the extent of conformational change reduced, particularly for fragile biomolecules.